Nitric oxide (NO) modulates several metabolic functions in hepatocytes, but the role of NO in bile secretion has not been clearly defined. In the present study, we examined the effects of NO on bile flow and biliary HCO3− and glutathione excretion in the isolated perfused rat liver and assessed the role of guanosine 3′,5′-cyclic monophosphate (cGMP) in mediating these effects. The NO donors sodium nitroprusside (SNP) and S-nitroso-acetyl-penicillamine stimulated bile flow and increased both HCO3− and glutathione excretion. Increases in bile flow were linearly related to increases in biliary glutathione concentration and output (P < .0001), which were almost entirely caused by glutathione disulfide, whereas the excretion of reduced glutathione remained unchanged. NO donors increased cGMP concentrations in bile and perfusate, and the membrane-permeant cGMP analogue dibutyryl cGMP was also found to stimulate bile flow and HCO3− excretion. However, in contrast to the NO donors, dibutyryl cGMP did not increase glutathione excretion. Furthermore, the NO donors failed to stimulate bile flow in mutant TR-rats in which the canalicular transport of glutathione and glutathione conjugates is deficient, although dibutyryl cGMP increased bile flow and HCO3− excretion in the mutant rats as in normals. These findings indicate that exogenous sources of NO increase bile acid-independent bile flow by stimulating glutathione disulfide excretion, effects that are independent of cGMP.